Search

Diabetes increases the likelihood of fracture, interferes with fracture healing and impairs angiogenesis. The latter may be significant due to the critical nature of angiogenesis in fracture healing. Although it is known that diabetes interferes with angiogenesis the mechanisms remain poorly defined. We examined fracture healing in normoglycemic and streptozotocin-induced diabetic mice and quantified the degree of angiogenesis with antibodies to three different vascular markers, CD34, CD31 and Factor VIII. The role of diabetes-enhanced inflammation was investigated by treatment of the TNFα-specific inhibitor, pegsunercept starting 10days after induction of fractures. Diabetes decreased both angiogenesis and VEGFA expression by chondrocytes. The reduced angiogenesis and VEGFA expression in diabetic fractures was rescued by specific inhibition of TNF in vivo. In addition, the TNF inhibitor rescued the negative effect of diabetes on endothelial cell proliferation and endothelial cell apoptosis. The effect of TNFα in vitro was enhanced by high glucose and an advanced glycation endproduct to impair microvascular endothelial cell proliferation and tube formation and to stimulate apoptosis. The effect of TNF, high glucose and an AGE was mediated by the transcription factor FOXO1, which increased expression of p21 and caspase-3. These studies indicate that inflammation plays a major role in diabetes-impaired angiogenesis in endochondral bone formation through its effect on microvascular endothelial cells and FOXO1.

Endochondral ossification is the process where cartilage forms prior to ossification and in which new bone forms during both fracture healing and ectopic bone formation. Transitioning to ossification is a highly coordinated process between hypertrophic chondrocytes, vascular endothelial cells, osteoblasts and osteoclasts. A critical biological process that is central to the interactions of these various cell types is angiogenesis. Although it is well established that angiogenesis is crucial for fracture repair, less is known pertaining to the role of angiogenesis in ectopic bone formation. Furthermore, fracture repair models are complicated by extensive trauma, subsequent inflammatory responses and concurrent repair processes in multiple tissues. In order to more definitively characterize the relationship between angiogenesis and postnatal endochondral ossification, a model of ectopic bone formation was used. Human demineralized bone matrix (DBM) was implanted in immune-deficient mice (rag null (B6.129S7-Rag1tm1/MOM/J)) to induce ectopic bone. Inhibition of angiogenesis with either a small molecule (TNP-470) or a targeted biological (Vascular Endothelial Growth Factor Receptor type 2 [VEGFR2] blocking antibody) prevented ectopic bone formation by 83% and 77%, respectively. Most striking was that the progression of chrondrogenesis was halted during very early phases of chondrocyte differentiation between condensation and prehypertrophy (TNP-470) or the proliferative phase (VEGFR2 blockade) prior to hypertrophy, while osteoclast recruitment and resorption were almost completely inhibited. Our results demonstrate angiogenesis plays a developmental role in endochondral bone formation at a much earlier phase of chondrogenesis than suggested by prior findings.

While the spatial activity of osteoblasts has been associated with modeling of bones during development, few studies have examined if variation in the spatial activity of osteoclasts also contributes to the morphogenesis of skeletal tissues. We examined this question by histomorphometric analysis and reconstructing the three-dimensional spatial distribution of osteoclasts in the femora of three inbred strains of male mice (A/J, C57BL/6J [B6], and C3H/HeJ [C3H]) that have differing skeletal, structural, and material properties. Our data show that total osteoclast surface area and osteoclast numbers are related to the overall bone density, but not related to the development of bone diameter or overall cortical area. The analysis of the spatial distribution of the osteoclasts showed that the asymmetrical mid-diaphyseal distribution of osteoclasts in A/J and B6 compared to the more uniform distribution of these cells around the circumference in the C3H mice was consistent with the more ellipsoid shape of A/J and B6 femora compared to the more circular mid-diaphyseal shape of the femora in the C3H mice. The statistically 2- to 3-fold fewer cells on the periosteal surface in the C3H compared to either the B6 or A/J mice is also consistent with the greater cortical thickness that is seen for the C3H mice compared to either B6 or A/J strains. In vitro studies of osteoclastogenesis and the expression of numerous phenotypic properties of osteoclasts prepared from the three strains of mice showed that A/J and B6 mice developed statistically greater numbers of tartrate resistant acid phosphatase (TRAP) positive cells and expressed statistically higher levels of multiple mRNAs that are unique to differentiated osteoclasts than those isolated from the C3H strain. In summary, the 3D reconstructions and histomorphometric analysis suggest that genetic differences lead to spatial variation in the distribution of osteoclasts. These variations in spatial distribution of osteoclasts in turn contribute in part to the development of the structural variations of the femora that are seen in the three strains of mice. In vitro studies suggest that intrinsic genetic variation in osteoclastogenesis and their phenotypic expression may contribute to the differences in their functional activities that give rise to the unique spatial distributions of these cells in bones.

Non-invasive characterization of fracture callus structure and composition may facilitate development of surrogate measures of the regain of mechanical function. As such, quantitative computed tomography- (CT-) based analyses of fracture calluses could enable more reliable clinical assessments of bone healing. Although previous studies have used CT to quantify and predict fracture healing, it is unclear which of the many CT-derived metrics of callus structure and composition are the most predictive of callus mechanical properties. The goal of this study was to identify the changes in fracture callus structure and composition that occur over time and that are most closely related to the regain of mechanical function. Micro-computed tomography (microCT) imaging and torsion testing were performed on murine fracture calluses (n=188) at multiple post-fracture timepoints and under different experimental conditions that alter fracture healing. Total callus volume (TV), mineralized callus volume (BV), callus mineralized volume fraction (BV/TV), bone mineral content (BMC), tissue mineral density (TMD), standard deviation of mineral density (sigma(TMD)), effective polar moment of inertia (J(eff)), torsional strength, and torsional rigidity were quantified. Multivariate statistical analyses, including multivariate analysis of variance, principal components analysis, and stepwise regression were used to identify differences in callus structure and composition among experimental groups and to determine which of the microCT outcome measures were the strongest predictors of mechanical properties. Although calluses varied greatly in the absolute and relative amounts of mineralized tissue (BV, BMC, and BV/TV), differences among timepoints were most strongly associated with changes in tissue mineral density. Torsional strength and rigidity were dependent on mineral density as well as the amount of mineralized tissue: TMD, BV, and sigma(TMD) explained 62% of the variation in torsional strength (p

Fracture healing involves multiple stages of repair and coordinated actions of multiple cell types. Consequently, it may be possible to enhance healing through treatment strategies that target more than one repair process or cell type. The goal of this study was to determine the combined effects of recombinant human bone morphogenetic protein 7 (rhBMP-7) and parathyroid hormone (PTH(1-34)) on metaphyseal bone healing. A wedge-shaped defect was created in the lateral aspect of the distal tibia in female New Zealand white rabbits (n=64) and was filled with tricalcium phosphate (TCP). Animals were assigned to four groups: 1) BMP-7 and PTH; 2) BMP-7; 3) PTH; and 4) control (TCP alone). In groups 1 and 2, 200 microg rhBMP-7 was incorporated into the TCP. Animals received daily subcutaneous injections of 10 microg/kg PTH(1-34) (groups 1 and 3) or saline (groups 2 and 4). Healing at 4 weeks was assessed using micro-computed tomography, histology, immunohistochemistry, and mechanical testing. Combined treatment with rhBMP-7 and PTH resulted in increased callus total volume (TV), mineralized volume (BV), average cross-sectional area, and bone mineral content (BMC) as compared to the control group (p

Recent studies from our laboratory demonstrate that TNF-alpha signaling contributes to the regulation of chondrocyte apoptosis and a lack of TNF-alpha signaling leads to a persistence of cartilaginous callus and delayed resorption of mineralized cartilage. This study examines how delays in the endochondral repair process affect the expression of specific mediators of proteolytic cartilage turnover and vascularization. Simple closed fractures were produced in wild type and TNF-alpha receptor (p55-/-/p75-/-)-deficient mice. Using ribonuclease protection assay (RPA) and microarray analysis, the expression of multiple mRNAs for various angiogenic factors and the metalloproteinase gene family were measured in fracture calluses. The direct actions of TNFalpha on the expression of specific angiogenic factors and metalloproteinases (MMPs) was examined in both cultured callus cells and articular chondrocytes to compare the effects of TNF-alpha in growth cartilage versus articular cartilage. MMPs 2, 9, 13, and 14 were quantitatively the most prevalent metalloproteases and all showed peaks in expression during the chondrogenic period. In the absence of TNF-alpha signaling, the expression of all of these mRNAs was reduced. The angiopoietin families of vascular regulators and their receptors were expressed at much higher levels than the VEGFs and their receptors and while the angiopoietins showed diminished or delayed expression in the absence of TNF-alpha signaling, VEGF and its receptors remained unaltered. The expression of vascular endothelial growth inhibitor (VEGI or TNFSF15) showed a near absence in its expression in the TNF-alpha receptor-deficient mice. In vitro assessment of cultured fracture callus cells in comparison to primary articular chondrocytes showed that TNF-alpha treatment specifically induced the expression of MMP9, MMP14, VEGI, and Angiopoietin 2. These results suggest that TNF-alpha signaling in chondrocytes controls vascularization of cartilage through the regulation of angiopoietin and VEGI factors which play counterbalancing roles in the induction of growth arrest, or apoptosis in endothelial cells. Furthermore, TNF-alpha appears to regulate, in part, the expression of two key proteolytic enzymes, MMP 9 and MMP14 that are known to be crucial to the progression of vascularization and turnover of mineralized cartilage. Thus, TNF-alpha signaling in healing fractures appears to coordinate the expression of specific regulators of endothelial cell survival and metalloproteolytic enzymes and is essential in the transition and progression of the endochondral phase of fracture repair.

Pleiotrophin (Ptn) is an extracellular matrix protein that regulates hippocampal synaptic plasticity and learning behavior in vivo. Since the overexpression of Ptn in transgenic mice leads to increased bone formation, we analyzed whether a deficiency in Ptn expression would have a negative effect on bone remodeling. Bones from Ptn-deficient mice and wild-type littermates were analyzed using radiography, muCT imaging and undecalcified histology. Biomechanical stability was determined in a three-point-bending assay. Cellular activities were assessed using dynamic histomorphometry and the determination of urinary collagen degradation products. Skeletons of Ptn-deficient mice have no gross abnormalities, displayed a normal size, and showed no differences in growth plate organization compared to wild-type littermates. There were no obvious differences in bone mass as determined by radiographic and muCT imaging. The absence of a bone remodeling phenotype in Ptn-deficient mice was further confirmed using static histomorphometry and biomechanical testing. Finally, the number, morphology, and function of osteoclasts, osteoblasts, and osteocytes were not altered in Ptn-deficient mice compared to wild-type littermates. The complete skeletal analysis of Ptn-deficient mice presented here demonstrates that the lack of Ptn in mice does not affect bone formation in vivo. Therefore, Ptn does not play a significant role in normal bone physiology.

Distraction osteogenesis (DO) is one of the most dramatic in vivo applications of mechanical stimulation as a means of inducing bone regeneration. A simple and reproducible murine model of tibia distraction osteogenesis was developed using a monolateral fixator. Bone formation was assessed histologically over a 35-day time course. The steady state expression of a broad family of angiogenesis-associated genes was assessed by microarray hybridization analyses over the same time course, while the immediate gene response that was induced during each cycle of distraction was assessed at 30 min and 8 h after the first and last rounds of activation of the fixator. Distraction osteogenesis promoted new bone formation primarily through an intramembranous process with maximal osteogenesis during the active distraction period. Histological analysis also showed that dense cortical bone continued to be formed, during the consolidation phase, for 2 weeks after distraction ended. The analysis of steady state mRNA expression levels over the time course of DO showed that VEGF-A and neuropilin, an alternate receptor for VEGF-A, both angiopoietin (Ang) 1 and 2 factors, and the Ang receptor Tie2 were the critical angiogenic factors during DO. A key transcriptional regulator of many of the angiogenic factors, hypoxia-induced factor1alpha (Hif-1a), the FGF binding protein pleiotropin/OSF1, and multiple MMP(s), were also induced during the active distraction period. Examination of the expression of angiogenic factors that were induced after each cycle of activation, demonstrated that Hif-1a, Nrp1, and VEGF-A were all cyclically induced after each increment of distraction. These results suggest that these factors are early mediators that are produced by distraction and contribute toward the processes that promote bone formation. These experiments represent the first step in defining the molecular mechanisms that regulate skeletal regeneration and the functional relationship between angiogenesis and osteogenesis during distraction osteogenesis.

The role of alpha-smooth muscle actin (SMA)-expressing fibroblasts in the contraction of skin wounds has been known for three decades. Recent studies have demonstrated that osteoblasts can also express the gene for this contractile muscle actin isoform and can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. These findings provided rationale for the hypothesis that SMA-expressing cells contribute to fracture healing by drawing the bone ends together. To begin to test this hypothesis, immunohistochemistry was employed to evaluate the distribution of connective tissue cells expressing SMA in a mouse model of successful fracture healing. The results demonstrated that the majority of the cells comprising the mesenchymal tissue interposed between the fracture ends contained SMA after 7 and 21 days, supporting the working hypothesis. Most of the osteoblasts lining the surfaces of newly forming bone and the chondrocytes comprising the cartilaginous callus also expressed this contractile actin isoform. The maximal SMA expression extended from 7 to 21 days postfracture. The finding of high levels of SMA expression in connective tissue cells participating in fracture healing suggests that SMA-enabled contraction may be playing a role in the healing process. These results warrant further study of the specific SMA-dependent cell behavior.

Vascular formation is intimately associated with bone formation during distraction osteogenesis (DO). While prior studies on this association have focused on vascular formation locally within the regenerate, we hypothesized that this vascular formation, as well as the resulting osteogenesis, relies heavily on the response of the vascular network in surrounding muscular compartments. To test this hypothesis, the spatiotemporal sequence of vascular formation was assessed in both muscular and osseous compartments in a murine model of DO and was compared to the progression of osteogenesis. Micro-computed tomography (μCT) scans were performed sequentially, before and after demineralization, on specimens containing contrast-enhanced vascular casts. Image registration and subtraction procedures were developed to examine the co-related, spatiotemporal patterns of vascular and osseous tissue formation. Immunohistochemistry was used to assess the contributory roles of arteriogenesis (formation of large vessels) and angiogenesis (formation of small vessels) to overall vessel formation. Mean vessel thickness showed an increasing trend during the period of active distraction (p=0.068), whereas vessel volume showed maximal increases during the consolidation period (p=0.009). The volume of mineralized tissue in the regenerate increased over time (p

The expression patterns of (bone morphogenetic proteins) BMPs during fracture repair and pre-natal bone development suggest that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) in culture provide a well recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs and to characterize their interactions. MSCs were harvested from the bone marrow of tibiae and femora of 8 to 10-week-old male C57/B6 mice and prepared by standard methods. Osteogenic differentiation was assessed by histological assays, alkaline phosphatase enzyme activity and assays for the expression of multiple mRNAs for BMPs and osteogenic development. The role of autogenously expressed BMPs in controlling the osteogenic differentiation of marrow stromal cells in vitro was assessed in both gain-of-function and loss-of-function experiments. Gain of function experiments were carried out in the presence of exogenously added BMP-2 or -7 and loss-of-function experiments were carried out by BMP antagonism with noggin and BMP-2 antibody blockade. Osteogenic differentiation was concurrent with and proportional to increases in the expression of BMPs-2, -3, -4, -5, -6 and -8A. BMP antagonism with either noggin or BMP-2 antibody blockade inhibited osteogenic differentiation by 50% to 80%, respectively, and reduced the expression of endogenous levels of BMPs-2, -3, -5 and -8A. In contrast, antagonism induced the expression of BMP-4 and -6. The addition of rhBMP-2 or -7 enhanced osteogenic differentiation and produced a reciprocal expression profile in the endogenous BMPs expression as compared to BMP antagonism. BMP antagonism could be rescued through the competitive addition of rhBMP-2. These studies demonstrated that osteogenic differentiation was regulated by a complex network of multiple BMPs that showed selective increased and decreased expression during differentiation. They further demonstrated that BMP-2 was a central regulator in this network.

Distraction osteogenesis is a unique and effective way to treat limb length inequality resulting from congenital and posttraumatic skeletal defects. However, despite its widespread clinical use, the cellular and molecular mechanisms by which this surgical treatment promotes new bone formation are not well understood. Previous studies in distraction osteogenesis have noted increased blood flow and vessel formation within the zone of distraction. These observations suggest that distraction osteogenesis may be driven in part by an angiogenic process. Using immunohistological analysis, the expression of two different angiogenic factors (VEGF and bFGF) was shown to localize at the leading edge of the distraction gap, where nascent osteogenesis was occurring. These cells were spatially adjacent to new vessels that were identified by staining for factor VIII. Microarray analysis detected maximal mRNA expression for a wide variety of angiogenic factors including angiopoietin 1 and 2, both Tie receptors, VEGF-A and -D, VEGFR2, and neuropilin 1. Expression of these factors was found to be maximal during the phase of active distraction. Expression of mRNA for extracellular matrix proteins and BMPs was also maximal during this period. A comparison between the patterns of gene expression in fracture healing and distraction osteogenesis revealed similarities; however, the expression of a number of genes showed selective expression in these two types of bone healing. These data suggest that bone formation during distraction osteogenesis is accompanied by the robust induction of factors associated with angiogenesis and support further investigations to elucidate the mechanisms by which angiogenic events promote bone repair and regeneration.